The present invention relates generally to the identification and isolation of novel DNA and to the recombinant production of novel polypeptides, designated herein as xe2x80x9cDcR3xe2x80x9d.
Various molecules, such as tumor necrosis factor-xcex1 (xe2x80x9cTNF-xcex1xe2x80x9d), tumor necrosis factor-xcex2 (xe2x80x9cTNF-xcex2xe2x80x9d or xe2x80x9clymphotoxinxe2x80x9d), CD30 ligand, CD27 ligand, CD40 ligand, OX-40 ligand, 4-1BB ligand, Fas ligand (also referred to as Apo-1 ligand or CD95 ligand), and Apo-2 ligand (also referred to as TRAIL) have been identified as members of the tumor necrosis factor (xe2x80x9cTNFxe2x80x9d) family of cytokines [See, e.g., Gruss and Dower, Blood, 85:3378-3404 (1995); Wiley et al., Immunity, 3:673-682 (1995); Pitti et al., J. Biol. Chem., 271:12687-12690 (1996)]. Among these molecules, TNF-xcex1, TNF-xcex2, CD30 ligand, 4-1BB ligand, Fas ligand, and Apo-2 ligand (TRAIL) have been reported to be involved in apoptotic cell death. Both TNF-xcex1 and TNF-xcex2 have been reported to induce apoptotic death in susceptible tumor cells [Schmid et al., Proc. Natl. Acad. Sci., 83:1881 (1986); Dealtry et al., Eur. J. Immunol., 17:689 (1987)]. Zheng et al. have reported that TNF-xcex1 is involved in post-stimulation apoptosis of CD8-positive T cells [Zheng et al., Nature, 377:348-351 (1995)]. Other investigators have reported that CD30 ligand may be involved in deletion of self-reactive T cells in the thymus [Amakawa et al., Cold Spring Harbor Laboratory Symposium on Programmed Cell Death, Abstr. No. 10, (1995)].
Fas ligand appears to regulate primarily three types of apoptosis: (a) activation-induced cell death (AICD) of mature T lymphocytes; (b) elimination of inflammatory cells from immune-privileged sites; and (c) killing of damaged cells by cytotoxic lymphocytes [Nagata, Cell, 88:355 (1997)]. It has been reported that T cell AICD assists in shutting down the host""s immune response once an infection has been cleared. Repeated stimulation of the T cell receptor (TCR) by antigen induced expression of Fas ligand and Fas on the surface of T helper cells; subsequently Fas ligand engages Fas and can trigger apoptosis in the activated lymphocytes, leading to their elimination. Immune-privileged sites include tissues such as the eye, brain or testis, in which inflammatory immune responses can perturb function. Cells in immune privileged sites appear to constitutively express Fas ligand, and eliminate infiltrating leukocytes that express Fas through Fas dependent apoptosis. Certain cancers including melanomas [Hahne et al., Science, 274:1363 (1996)] and hepatocellular carcinomas [Strand et al., Nature Med., 2:1361-1366 (1996)] use a similar Fas ligand-dependent mechanism to evade immune survaillance. Natural killer (NK) cells and cytotoxic T lymphocytes have been reported to eliminate cells that have been damaged by viral or bacterial infection or by oncogenic transformation by at least two pathways. One pathway involves release of perforin and granzymes, and an alternative pathway involves expression of Fas ligand and induction of apoptosis by engagement of Fas on target cells [Nagata, supra; Moretta, Cell, 90:13 (1997)].
Mutations in the mouse Fas/Apo-1 receptor or ligand genes (called 1pr and gld, respectively) have been associated with some autoimmune disorders, indicating that Fas ligand may play a role in regulating the clonal deletion of self-reactive lymphocytes in the periphery [Krammer et al., Curr. Op. Immunol., 6:279-289 (1994); Nagata et al., Science, 267:1449-1456 (1995)]. Fas ligand is also reported to induce post-stimulation apoptosis in CD4-positive T lymphocytes and in B lymphocytes, and may be involved in the elimination of activated lymphocytes when their function is no longer needed [Krammer et al., supra; Nagata et al., supra]. Agonist mouse monoclonal antibodies specifically binding to the Fas receptor have been reported to exhibit cell killing activity that is comparable to or similar to that of TNF-xcex1 [Yonehara et al., J. Exp. Med., 169:1747-1756 (1989)].
Induction of various cellular responses mediated by such TNF family cytokines is believed to be initiated by their binding to specific cell receptors. Two distinct TNF receptors of approximately 55-kDa (TNFR1) and 75-kDa (TNFR2) have been identified [Hohmann et al., J. Biol. Chem., 264:14927-14934 (1989); Brockhaus et al., Proc. Natl. Acad. Sci., 87:3127-3131 (1990); EP 417,563, published Mar. 20, 1991] and human and mouse cDNAs corresponding to both receptor types have been isolated and characterized [Loetscher et al., Cell, 61:351 (1990); Schall et al., Cell, 61:361 (1990); Smith et al., Science, 248:1019-1023 (1990); Lewis et al., Proc. Natl. Acad. Sci., 88:2830-2834 (1991); Goodwin et al., Mol. Cell. Biol., 11:3020-3026 (1991)]. Extensive polymorphisms have been associated with both TNF receptor genes [see, e.g., Takao et al., Immunogenetics, 37:199-203 (1993)]. Both TNFRs share the typical structure of cell surface receptors including extracellular, transmembrane and intracellular regions. The extracellular portions of both receptors are found naturally also as soluble TNF-binding proteins [Nophar, Y. et al., EMBO J., 9:3269 (1990); and Kohno, T. et al., Proc. Natl. Acad. Sci. U.S.A., 87:8331 (1990)]. More recently, the cloning of recombinant soluble TNF receptors was reported by Hale et al. [J. Cell. Biochem. Supplement 15F, 1991, p. 113 (P424)].
The extracellular portion of type 1 and type 2 TNFRs (TNFR1 and TNFR2) contains a repetitive amino acid sequence pattern of four cysteine-rich domains (CRDs) designated 1 through 4, starting from the NH2-terminus. Each CRD is about 40 amino acids long and contains 4 to 6 cysteine residues at positions which are well conserved [Schall et al., supra; Loetscher et al., supra; Smith et al., supra; Nophar et al., supra; Kohno et al., supra]. In TNFR1, the approximate boundaries of the four CRDs are as follows: CRD1-amino acids 14 to about 53; CRD2-amino acids from about 54 to about 97; CRD3-amino acids from about 98 to about 138; CRD4-amino acids from about 139 to about 167. In TNFR2, CRD1 includes amino acids 17 to about 54; CRD2-amino acids from about 55 to about 97; CRD3-amino acids from about 98 to about 140; and CRD4-amino acids from about 141 to about 179 [Banner et al., Cell, 73:431-445 (1993)]. The potential role of the CRDs in ligand binding is also described by Banner et al., supra.
A similar repetitive pattern of CRDs exists in several other cell-surface proteins, including the p75 nerve growth factor receptor (NGFR) [Johnson et al., Cell, 47:545 (1986); Radeke et al., Nature, 325:593 (1987)], the B cell antigen CD40 [Stamenkovic et al., EMBO J., 8:1403 (1989)], the T cell antigen OX40 [Mallett et al., EMBO J., 9:1063 (1990)] and the Fas antigen [Yonehara et al., supra and Itoh et al., Cell, 66:233-243 (1991)]. CRDs are also found in the soluble TNFR (sTNFR)-like T2 proteins of the Shope and myxoma poxviruses [Upton et al., Virology, 160:20-29 (1987); Smith et al., Biochem. Biophys. Res. Commun., 176:335 (1991); Upton et al., Virology, 184:370 (1991)]. Optimal alignment of these sequences indicates that the positions of the cysteine residues are well conserved. These receptors are sometimes collectively referred to as members of the TNF/NGF receptor superfamily. Recent studies on p75NGFR showed that the deletion of CRD1 [Welcher, A. A. et al., Proc. Natl. Acad. Sci. USA, 88:159-163 (1991)] or a 5-amino acid insertion in this domain [Yan, H. and Chao, M. V., J. Biol. Chem., 266:12099-12104 (1991)] had little or no effect on NGF binding [Yan, H. and Chao, M. V., supra]. p75 NGFR contains a proline-rich stretch of about 60 amino acids, between its CRD4 and transmembrane region, which is not involved in NGF binding [Peetre, C. et al., Eur. J. Haematol., 41:414-419 (1988); Seckinger, P. et al., J. Biol. Chem., 264:11966-11973 (1989); Yan, H. and Chao, M. V., supra]. A similar proline-rich region is found in TNFR2 but not in TNFR1.
Itoh et al. disclose that the Fas receptor can signal an apoptotic cell death similar to that signaled by the 55-kDa TNFR1 [Itoh et al., supra]. Expression of the Fas antigen has also been reported to be down-regulated along with that of TNFR1 when cells are treated with either TNF-xcex1 or anti-Fas mouse monoclonal antibody [Krammer et al., supra; Nagata et al., supra]. Accordingly, some investigators have hypothesized that cell lines that co-express both Fas and TNFR1 receptors may mediate cell killing through common signaling pathways [Id.].
The TNF family ligands identified to date, with the exception of lymphotoxin-xcex1, are type II transmembrane proteins, whose C-terminus is extracellular. In contrast, most receptors in the TNF receptor (TNFR) family identified to date are type I transmembrane proteins. In both the TNF ligand and receptor families, however, homology identified between family members has been found mainly in the extracellular domain (xe2x80x9cECDxe2x80x9d). Several of the TNF family cytokines, including TNF-xcex1, Fas ligand and CD40 ligand, are cleaved proteolytically at the cell surface; the resulting protein in each case typically forms a homotrimeric molecule that functions as a soluble cytokine. TNF receptor family proteins are also usually cleaved proteolytically to release soluble receptor ECDs that can function as inhibitors of the cognate cytokines.
Recently, other members of the TNFR family have been identified. Such newly identified members of the TNFR family include CAR1, HVEM and osteoprotegerin (OPG) [Brojatsch et al., Cell, 87:845-855 (1996); Montgomery et al., Cell, 87:427-436 (1996); Marsters et al., J. Biol. Chem., 272:14029-14032 (1997); Simonet et al., Cell, 89:309-319 (1997)]. Unlike other known TNFR-like molecules, Simonet et al., supra, report that OPG contains no hydrophobic transmembrane-spanning sequence.
In Marsters et al., Curr. Biol., 6:750 (1996), investigators describe a full length native sequence human polypeptide, called Apo-3, which exhibits similarity to the TNFR family in its extracellular cysteine-rich repeats and resembles TNFR1 and CD95 in that it contains a cytoplasmic death domain sequence [see also Marsters et al., Curr. Biol., 6:1669 (1996)]. Apo-3 has also been referred to by other investigators as DR3, wsl-1 and TRAMP [Chinnaiyan et al., Science, 274:990 (1996); Kitson et al., Nature, 384:372 (1996); Bodmer et al., Immunity, 6:79 (1997)].
Pan et al. have disclosed another TNF receptor family member referred to as xe2x80x9cDR4xe2x80x9d [Pan et al., Science, 276:111-113 (1997)]). The DR4 was reported to contain a cytoplasmic death domain capable of engaging the cell suicide apparatus. Pan et al. disclose that DR4 is believed to be a receptor for the ligand known as Apo-2 ligand or TRAIL.
In Sheridan et al., Science, 277:818-821 (1997) and Pan et al., Science, 277:815-818 (1997), another molecule believed to be a receptor for the Apo-2 ligand (TRAIL) is described. That molecule is referred to as DR5 (it has also been alternatively referred to as Apo-2). Like DR4, DR5 is reported to contain a cytoplasmic death domain and be capable of signaling apoptosis.
In Sheridan et al., supra, a receptor called DcR1 (or alternatively, Apo-2DcR) is disclosed as being a potential decoy receptor for Apo-2 ligand (TRAIL). Sheridan et al. report that DcR1 can inhibit Apo-2 ligand function in vitro. See also, Pan et al., supra, for disclosure on the decoy receptor referred to as TRID.
For a review of the TNF family of cytokines and their receptors, see Gruss and Dower, supra.
Membrane-bound proteins and receptors can play an important role in the formation, differentiation and maintenance of multicellular organisms. The fate of many individual cells, e.g., proliferation, migration, differentiation, or interaction with other cells, is typically governed by information received from other cells and/or the immediate environment. This information is often transmitted by secreted polypeptides (for instance, mitogenic factors, survival factors, cytotoxic factors, differentiation factors, neuropeptides, and hormones) which are, in turn, received and interpreted by diverse cell receptors or membrane-bound proteins. Such membrane-bound proteins and cell receptors include, but are not limited to, cytokine receptors, receptor kinases, receptor phosphatases, receptors involved in cell-cell interactions, and cellular adhesin molecules like selectins and integrins. For instance, transduction of signals that regulate cell growth and differentiation is regulated in part by phosphorylation of various cellular proteins. Protein tyrosine kinases, enzymes that catalyze that process, can also act as growth factor receptors. Examples include fibroblast growth factor receptor and nerve growth factor receptor.
Membrane-bound proteins and receptor molecules have various industrial applications, including as pharmaceutical and diagnostic agents. Receptor immunoadhesins, for instance, can be employed as therapeutic agents to block receptor-ligand interaction. The membrane-bound proteins can also be employed for screening of potential peptide or small molecule inhibitors of the relevant receptor/ligand interaction.
Efforts are being undertaken by both industry and academia to identify new, native receptor proteins. Many efforts are focused on the screening of mammalian recombinant DNA libraries to identify the coding sequences for novel receptor proteins.
Applicants have identified a cDNA clone that encodes a novel polypeptide, designated in the present application as xe2x80x9cDcR3.xe2x80x9d The term xe2x80x9cDcR3xe2x80x9d as used herein refers to the same polypeptides previously referred to by Applicants as xe2x80x9cDNA30942xe2x80x9d.
In one embodiment, the invention provides an isolated nucleic acid molecule comprising DNA encoding DcR3 polypeptide. In one aspect, the isolated nucleic acid comprises DNA encoding DcR3 polypeptide having amino acid residues 1 to 300 of FIG. 1 (SEQ ID NO:1); residues 1 to 215 of FIG. 1 (SEQ ID NO:1); or residues 1 to x, where x is any one of residues 215 to 300 of FIG. 1 (SEQ ID NO:1), or is complementary to such encoding nucleic acid sequence, and remains stably bound to it under at least moderate, and optionally, under high stringency conditions.
In another embodiment, the invention provides a vector comprising DNA encoding DcR3 polypeptide. A host cell comprising such a vector is also provided. By way of example, the host cells may be CHO cells, E. coli, or yeast. A process for producing DcR3 polypeptides is further provided and comprises culturing host cells under conditions suitable for expression of DcR3 and recovering DcR3 from the cell culture.
In another embodiment, the invention provides isolated DcR3 polypeptide. In particular, the invention provides isolated native sequence DcR3 polypeptide, which in one embodiment, includes an amino acid sequence comprising residues 1 to 300 of FIG. 1 (SEQ ID NO:1) or residues 1 to 215 of FIG. 1 (SEQ ID NO:1) or residues 1 to x, where x is any one of residues 215 to 300 of FIG. 1 (SEQ ID NO:1).
In another embodiment, the invention provides isolated DcR3 variants. The DcR3 variants comprise polypeptides which have at least about 80% amino acid sequence identity with the deduced amino acid sequence of FIG. 1 (SEQ ID NO:1) or domain sequences identified herein, and preferably have activity(s) of native or naturally-occurring DcR3.
In another embodiment, the invention provides chimeric molecules comprising DcR3 polypeptide fused to a heterologous polypeptide or amino acid sequence. An example of such a chimeric molecule comprises a DcR3 fused to an epitope tag sequence or a Fc region of an immunoglobulin.
In another embodiment, the invention provides an antibody which specifically binds to DcR3 polypeptide. Optionally, the antibody is a monoclonal antibody. Optionally, the antibody is a monoclonal antibody which specifically binds to DcR3 and blocks its binding to Fas ligand and/or other ligands recognized by DcR3.
In a further embodiment, the invention provides agonists and antagonists of DcR3 polypeptide. Therapeutic and diagnostic methods are also provided.
In another embodiment, the invention provides an expressed sequence tag (EST) comprising the nucleotide sequence of FIG. 3 (SEQ ID NO:3).